scholarly journals Insulating DNA directs ubiquitous transcription of the Drosophila melanogaster alpha 1-tubulin gene.

1994 ◽  
Vol 14 (9) ◽  
pp. 6398-6408 ◽  
Author(s):  
K H O'Donnell ◽  
C T Chen ◽  
P C Wensink
Keyword(s):  
Drosophila Melanogaster ◽  
Developmental Stages ◽  
Germ Line ◽  
Core Promoter ◽  
High Sensitivity ◽  
In Vitro Transcription ◽  
Gene Products ◽  
Position Effects ◽  
Germ Line Transformation

We identify DNA regions that are necessary for the ubiquitous expression of the Drosophila melanogaster alpha 1-tubulin (alpha 1t) gene. In vitro transcription showed that two upstream regions, tubulin element 1 (TE1 [29 bp]) and tubulin element 2 (TE2 [68 bp]), and a downstream region activate transcription. Germ line transformation demonstrated that these three regions are sufficient to direct the alpha 1t core promoter to begin transcribing at the stage of cellular blastoderm formation and to continue thereafter at high levels in all tissues and developmental stages. Remarkably, mutation of any one of these regions results in high sensitivity to chromosomal position effects, producing different but reproducible tissue-specific patterns of expression in each transformed line. None of these regions behaves as an enhancer in a conventional germ line transformation test. These observations show that these three regions, two of which bind the GAGA transcription factor, act ubiquitously to insulate from position effects and to activate transcription. The results also provide vectors for ubiquitous expression of gene products and for examining silencer activities.

Insulating DNA directs ubiquitous transcription of the Drosophila melanogaster alpha 1-tubulin gene

1994 ◽  
Vol 14 (9) ◽  
pp. 6398-6408
Author(s):  
K H O'Donnell ◽  
C T Chen ◽  
P C Wensink
Keyword(s):  
Drosophila Melanogaster ◽  
Developmental Stages ◽  
Germ Line ◽  
Core Promoter ◽  
High Sensitivity ◽  
In Vitro Transcription ◽  
Gene Products ◽  
Position Effects ◽  
Germ Line Transformation

We identify DNA regions that are necessary for the ubiquitous expression of the Drosophila melanogaster alpha 1-tubulin (alpha 1t) gene. In vitro transcription showed that two upstream regions, tubulin element 1 (TE1 [29 bp]) and tubulin element 2 (TE2 [68 bp]), and a downstream region activate transcription. Germ line transformation demonstrated that these three regions are sufficient to direct the alpha 1t core promoter to begin transcribing at the stage of cellular blastoderm formation and to continue thereafter at high levels in all tissues and developmental stages. Remarkably, mutation of any one of these regions results in high sensitivity to chromosomal position effects, producing different but reproducible tissue-specific patterns of expression in each transformed line. None of these regions behaves as an enhancer in a conventional germ line transformation test. These observations show that these three regions, two of which bind the GAGA transcription factor, act ubiquitously to insulate from position effects and to activate transcription. The results also provide vectors for ubiquitous expression of gene products and for examining silencer activities.

The ovarian, ecdysterone, and heat-shock-responsive promoters of the Drosophila melanogaster hsp27 gene react very differently to perturbations of DNA sequence

1987 ◽  
Vol 7 (3) ◽  
pp. 973-981
Author(s):  
E P Hoffman ◽  
S L Gerring ◽  
V G Corces
Keyword(s):  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Dna Sequence ◽  
Germ Line ◽  
P Element ◽  
Position Effects ◽  
Consensus Sequences ◽  
Basal Expression ◽  
Upstream Promoter ◽  
Germ Line Transformation

The effect of various types of DNA sequence alterations on the activity of the ovarian, ecdysterone, and heat-shock-responsive promoters of the Drosophila melanogaster hsp27 gene was studied by P element-mediated germ line transformation. Regions of DNA required for proper expression of the gene under these different conditions were identified. Wild-type levels of transcription during oogenesis are dependent on two elements respectively located within a 64-base-pair (bp) fragment in the transcribed untranslated region and between -227 and -958 bp upstream of the transcription start site. This ovarian expression is particularly sensitive to both chromosomal position effects and an increased distance between the distal upstream promoter element and the TATAA homology. The ecdysterone-mediated expression during metamorphosis is dependent on a 145-bp domain including the TATAA box and additional upstream sequences that augment transcription by two- to five-fold. Finally, sequences necessary for heat shock expression are located much further upstream from hsp27 than those previously found for hsp70, although basal expression was correlated with the presence of more proximal heat shock consensus sequences.

scholarly journals The ovarian, ecdysterone, and heat-shock-responsive promoters of the Drosophila melanogaster hsp27 gene react very differently to perturbations of DNA sequence.

1987 ◽  
Vol 7 (3) ◽  
pp. 973-981 ◽  
Author(s):  
E P Hoffman ◽  
S L Gerring ◽  
V G Corces
Keyword(s):  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Dna Sequence ◽  
Germ Line ◽  
P Element ◽  
Position Effects ◽  
Consensus Sequences ◽  
Basal Expression ◽  
Upstream Promoter ◽  
Germ Line Transformation

The effect of various types of DNA sequence alterations on the activity of the ovarian, ecdysterone, and heat-shock-responsive promoters of the Drosophila melanogaster hsp27 gene was studied by P element-mediated germ line transformation. Regions of DNA required for proper expression of the gene under these different conditions were identified. Wild-type levels of transcription during oogenesis are dependent on two elements respectively located within a 64-base-pair (bp) fragment in the transcribed untranslated region and between -227 and -958 bp upstream of the transcription start site. This ovarian expression is particularly sensitive to both chromosomal position effects and an increased distance between the distal upstream promoter element and the TATAA homology. The ecdysterone-mediated expression during metamorphosis is dependent on a 145-bp domain including the TATAA box and additional upstream sequences that augment transcription by two- to five-fold. Finally, sequences necessary for heat shock expression are located much further upstream from hsp27 than those previously found for hsp70, although basal expression was correlated with the presence of more proximal heat shock consensus sequences.

In vitro transcription and translation of simian rotavirus SA11 gene products.

1980 ◽  
Vol 33 (3) ◽  
pp. 1111-1121 ◽  
Author(s):  
B B Mason ◽  
D Y Graham ◽  
M K Estes
Keyword(s):  
In Vitro Transcription ◽  
Gene Products

Delimiting regulatory sequences of the Drosophila melanogaster Ddc gene

1986 ◽  
Vol 6 (12) ◽  
pp. 4548-4557
Author(s):  
J Hirsh ◽  
B A Morgan ◽  
S B Scholnick
Keyword(s):  
Drosophila Melanogaster ◽  
Germ Line ◽  
Regulatory Elements ◽  
P Element ◽  
Developmental Expression ◽  
Upstream Region ◽  
Regulatory Sequences ◽  
Flanking Sequences

We delimited sequences necessary for in vivo expression of the Drosophila melanogaster dopa decarboxylase gene Ddc. The expression of in vitro-altered genes was assayed following germ line integration via P-element vectors. Sequences between -209 and -24 were necessary for normally regulated expression, although genes lacking these sequences could be expressed at 10 to 50% of wild-type levels at specific developmental times. These genes showed components of normal developmental expression, which suggests that they retain some regulatory elements. All Ddc genes lacking the normal immediate 5'-flanking sequences were grossly deficient in larval central nervous system expression. Thus, this upstream region must contain at least one element necessary for this expression. A mutated Ddc gene without a normal TATA boxlike sequence used the normal RNA start points, indicating that this sequences is not required for start point specificity.

scholarly journals Translation initiation in Drosophila melanogaster is reduced by mutations upstream of the AUG initiator codon.

1991 ◽  
Vol 11 (4) ◽  
pp. 2149-2153 ◽  
Author(s):  
Y Feng ◽  
L E Gunter ◽  
E L Organ ◽  
D R Cavener
Keyword(s):  
Translation Initiation ◽  
Larval Stage ◽  
Developmental Stages ◽  
Germ Line ◽  
Adult Stage ◽  
Mutant Gene ◽  
Start Codon ◽  
Fold Reduction

The importance to in vivo translation of sequences immediately upstream of the Drosophila alcohol dehydrogenase (Adh) start codon was examined at two developmental stages. Mutations were introduced into the Adh gene in vitro, and the mutant gene was inserted into the genome via germ line transformation. An A-to-T substitution at the -3 position did not affect relative translation rates of the ADH protein at the second-instar larval stage but resulted in a 2.4-fold drop in translation of ADH at the adult stage. A second mutant gene, containing five mutations in the region -1 to -9, was designed to completely block translation initiation. However, transformant lines bearing these mutations still exhibit detectable ADH, albeit at substantially reduced levels. The average fold reduction at the second-instar larval stage was 5.9, while at the adult stage a 12.5-fold reduction was observed.

Transformation mapping of the regulatory elements of the ecdysone-inducible P1 gene of Drosophila melanogaster

1991 ◽  
Vol 11 (5) ◽  
pp. 2913-2917
Author(s):  
F Maschat ◽  
M L Dubertret ◽  
J A Lepesant
Keyword(s):  
Drosophila Melanogaster ◽  
Hormonal Regulation ◽  
Germ Line ◽  
Regulatory Elements ◽  
Fat Bodies ◽  
Germ Line Transformation ◽  
Third Instar Larvae

The transcription of the P1 gene is induced by 20-hydroxyecdysone in fat bodies of third-instar larvae. Germ line transformation showed that sequences between -138 to +276 contain elements required for a qualitatively correct developmental and hormonal regulation of P1 transcription. Sequences from -138 to -68 are essential for this expression.

Calreticulin Mediates Anesthetic Sensitivity in Drosophila melanogaster 

2003 ◽  
Vol 99 (4) ◽  
pp. 867-875 ◽  
Author(s):  
Sumiko Gamo ◽  
Junya Tomida ◽  
Katsuyuki Dodo ◽  
Dai Keyakidani ◽  
Hitoshi Matakatsu ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Double Mutant ◽  
Developmental Stages ◽  
Northern Blotting ◽  
General Anesthetics ◽  
Wild Type ◽  
Mutant Strains ◽  
Low Expression

Background Various species, e.g., Caenorhabditis elegans, Drosophila melanogaster, and mice, have been used to explore the mechanisms of action of general anesthetics in vivo. The authors isolated a Drosophila mutant, ethas311, that was hypersensitive to diethylether and characterized the calreticulin (crc) gene as a candidate of altered anesthetic sensitivity. Methods Molecular analysis of crc included cloning and sequencing of the cDNA, Northern blotting, and in situ hybridization to accomplish the function of the gene and its mutation. For anesthetic phenotype assay, the 50% anesthetizing concentrations were determined for ethas311, revertants, and double-mutant strains (wild-type crc transgene plus ethas311). Results Expression of the crc 1.4-kb transcript was lower in the mutant ethas311 than in the wild type at all developmental stages. The highest expression at 19 h after pupation was observed in the brain of the wild type but was still low in the mutant at that stage. The mutant showed resistance to isoflurane as well as hypersensitivity to diethylether, whereas it showed the wild phenotype to halothane. Both mutant phenotypes were restored to the wild type in the revertants and double-mutant strains. Conclusion ethas311 is a mutation of low expression of the Drosophila calreticulin gene. The authors demonstrated that hypersensitivity to diethylether and resistance to isoflurane are associated with low expression of the gene. In Drosophila, calreticulin seems to mediate these anesthetic sensitivities, and it is a possible target for diethylether and isoflurane, although the predicted anesthetic targets based on many studies in vitro and in vivo are the membrane proteins, such as ion channels and receptors.

scholarly journals Solution NMR readily reveals distinct structural folds and interactions in doubly 13C- and 19F-labeled RNAs

2020 ◽  
Vol 6 (41) ◽  
pp. eabc6572
Author(s):  
Owen B. Becette ◽  
Guanghui Zong ◽  
Bin Chen ◽  
Kehinde M. Taiwo ◽  
David A. Case ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Sensitivity ◽  
Binding Pocket ◽  
In Vitro Transcription ◽  
Rapid Identification ◽  
Spin Pair ◽  
Encapsidation Signal ◽  
Spin Pairs ◽  
Critical Components

RNAs form critical components of biological processes implicated in human diseases, making them attractive for small-molecule therapeutics. Expanding the sites accessible to nuclear magnetic resonance (NMR) spectroscopy will provide atomic-level insights into RNA interactions. Here, we present an efficient strategy to introduce 19F-13C spin pairs into RNA by using a 5-fluorouridine-5′-triphosphate and T7 RNA polymerase–based in vitro transcription. Incorporating the 19F-13C label in two model RNAs produces linewidths that are twice as sharp as the commonly used 1H-13C spin pair. Furthermore, the high sensitivity of the 19F nucleus allows for clear delineation of helical and nonhelical regions as well as GU wobble and Watson-Crick base pairs. Last, the 19F-13C label enables rapid identification of a small-molecule binding pocket within human hepatitis B virus encapsidation signal epsilon (hHBV ε) RNA. We anticipate that the methods described herein will expand the size limitations of RNA NMR and aid with RNA-drug discovery efforts.

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